The discovery and development of fundamentally new classes of antibiotics to treat bacterial infections has slowed dramatically. This is a critica problem to human health due to the emergence multi-drug resistant bacterial strains, which present a threat to both hospitals and communities at large. The long-term goal is to create new methods for studying the mechanisms leading to efficacy or resistance, while developing antibiotics that can overcome resistance. This application's objective is to site-specifically incorporate unnatural amino acids into ribosomally synthesized thiopeptide antibiotics produced by the soil bacteria, Streptomyces. The central hypothesis is that ribosomal incorporation of these unnatural amino acids will create a rapid and general method to study existing and future thiopeptide mechanisms of action, as well as drastically improve the pharmacological properties of thiopeptides for human clinical use. This contrasts with previous synthetic strategies, which have been hindered by the high molecular complexity of thiopeptides. The central hypothesis is based on the demonstrated utility of unnatural amino acids for the creation of biophysical and cell biological probes, biologically active peptide and antibody scaffolds, and improved medicinal bioconjugates. The rationale for this proposal is that understanding novel antibiotic thiopeptide mechanisms and creation of new thiopeptide antibiotics scaffolds has the potential to translate into new medicines to treat the roughly 2 million cases of drug-resistant bacterial infections reported each year. The central hypothesis will be tested with the following specific aims: 1) Elucidate berninamycin's antibiotic mechanism of action and 2) Identify improved analogues of thiopeptide GE37468. Insertion of unnatural amino acids into thiopeptides berninamycin will allow labeling studies to test the hypothesis that it possess a unique dual mode of action, and modification of GE37468's core scaffold will generate improved thiopeptides to test against multi-drug resistant bacterial strains. Incorporation of unnatural amino acids will be accomplished using established orthogonal aminoacyl- tRNA synthetase (aaRS)/tRNA pairs and known thiopeptide producing gene clusters into Streptomycetes. This approach is innovative because it combines both existing biosynthetic ribosomal machinery with chemical synthesis (unnatural amino acids) to produce thiopeptides with precisely engineered structure and reactivity. Significantly, this research stands to increase our understanding of novel antibiotic mechanisms, and create a new model for the creation of thiopeptide antibiotic analogues.